One of the most important factors to be considered in developing a plant transformation procedure is the availability of a promoter which provides reliable high level expression of introduced genes in the target cells. For example, for the transformation of plant cells with DNA encoding an antibiotic resistance marker, it is clearly desirable to obtain a high level of expression of the introduced gene to enable efficient selection of transformants. Moreover, in cases where the untransformed tissue shows a degree of natural resistance to the antibiotic, e.g., wheat and maize embryo tissue selected on kanamycin (Hauptmann et al. (1988) Plant Physiol. 86:602-606), a strong selection system would be critical for the successful production of transformed plants.
Promoters are the portions of DNA sequence at the beginnings of genes which contain the signals for RNA polymerase to begin transcription so that protein synthesis can then proceed. Eukaryotic promoters are complex, and are comprised of components which include a TATA box consensus sequence at about 35 bp 5' relative to the transcription start site, or cap site, which is defined as +1 (Breathnach and Chambon (1981) Ann. Rev. Biochem. 50:349-383; Messing et al. (1983) in Genetic Engineering of Plants, T. Kosuge, Meredith and Hollaender, (eds.), pp.211-227). In most instances the TATA box is required for accurate transcription initiation. Further upstream, often between -80 and -100, there can be a promoter element with homology to the consensus sequence CCAAT (Breathnach and Chambon (1981) supra. In plants the CCAAT box may be substituted by a consensus sequence which Messing et al. (1983) have termed the AGGA box, positioned a similar distance from the cap site. Additional DNA sequence in the 5' untranscribed region are believed to be involved in the modulation of gene expression. There are DNA sequences which affect gene expression in response to environmental stimuli, such as illumination or nutrient availability or adverse conditions including heat shock, anaerobiosis, or the presence of heavy metals. There are also DNA sequences which control gene expression during development, or in a tissue-specific fashion. Other DNA sequences have been found to elevate the overall level of expression of the nearby genes; such sequences have been termed "enhancers" in animal and plant systems. In yeast, similar stimulatory sequences are known which are called "upstream activating sequences," which also often appear to carry regulatory information. Promoters are usually positioned 5', or upstream, relative to the start of the coding region of the corresponding gene, and the tract containing all the ancillary elements affecting regulation or absolute levels of transcription may be comprised of less than 100 bp or as much as 1 kbp.
Among promoters that have been widely used in plant cell transformations are those of two genes encoding alcohol dehydrogenase, Adh1 and Adh2. Both Adh1 and Adh2 are induced after the onset of anaerobiosis (Freeling (1973) Mol. Gen. Genet. 127:215-227). Of the two enzymes, Adh1 is the one of primary importance during anaerobic conditions (Freeling et al. (1973) Biochem. Genet. 8:27-23). Maize Adh1 has been cloned and sequenced (dennis et al. (1984) Nucl. Acids Res. 12:3983-4000) as has been Adh2 (Dennis et al. (1985) Nucl. Acids Res. 13:727-743). Adh1 genes from other sources have also recently been cloned and sequenced (Llewellyn et al. (1987) J. Mol. Biol. 195:115-123). Howard et al. (1987) Planta 170:535-540 examined the expression of the endogenous Adh1 gene and a chimeric Adh1 gene in maize protoplasts. The Adh1 chimeric gene ADH-CAT consists of the Adh1 promoter linked to the chloramphenicol acetyltransferase (CAT) coding sequences and nopaline synthase (nos) 3' signal. ADH-CAT, introduced into maize protoplasts by electroporation, was expressed approximately four-fold higher at low oxygen concentrations than under control conditions. Expression of ADH-CAT paralleled the expression of the endogenous Adh1 gene in maize protoplasts and the anaerobic response in cell culture was qualitatively similar to the response in maize seedlings. Walker et al. (1987) Proc. Natl. Acad. Sci. 84:6624-6628 identified the sequence elements necessary for anaerobic induction of ADH-CAT based on the expression of a series of in vitro manipulated ADH-CAT chimeric genes. They showed that there is an anaerobic regulatory element (ARE) between positions -140 and -99 of the maize Adh1 promoter, and that the ARE is composed of at least two sequence elements, positions -133 to -124 and positions -113 to -99, both of which are necessary, and together are sufficient for low oxygen expression of ADH-CAT gene activity.
It was further reported (Walker et al. (1987) supra) that the Adh2 gene of maize is also regulated by anaerobiosis and contains homology to the Adh1 ARE. The homology is approximately 81% in Region I of the ARE and approximately 69% in Region II. Also, the 5'-flanking regions of the Adh genes from Arabidoosis and pea were reported to be not greater than 60% homologous to the maize Adh1 ARE over a 10 bp region.
The 35S promoter of Cauliflower Mosaic Virus (Guilley et al. (1982) Cell 30:763-773; Odell et al. (1985) supra) is one of the most frequently used promoters in plant transformation procedures. This dicot virus promoter directs expression of genes introduced into protoplasts of dicots and monocots (Fromm et al. (1985) Proc. Natl. Acad. Sci 82:5824-5828; Nagata et al. (1987) Mol. Gen. Genet. 207:242-244; Odell et al. (1988) Plant Mol. Biol. 10:263-273). Quantitative measurements of relative transcript levels in transformed tobacco cells (Morelli et al. (1985) Nature 315:200-204; Nagy et al. (1985), in Biotechnology in Plant Science: Relevance to Agriculture in the Eighties, M. Zaitlin, P. Day, and A. Hollaender, (eds.), Academic Press, New York, pp. 227-236) or transgenic petunia plants (Sanders et al. (1987) Nucl. Acids Res. 15:1543-1558) showed that the 35S promoter was at least 30 times stronger than the nos promoter. The strength of the 35S promoter accounts for its widespread use for high level expression of desirable traits in transgenic plants. Fang et al. (1989) The Plant Cell 1:141-150 have shown by 5', 3', and internal deletions that the -343 to -46 upstream fragment can be subdivided into three functional regions, -343 to -208, -208 to 31 90, and -90 to -46. They showed that the first two regions potentiated transcriptional activity when tested with the appropriate 35S promoter sequence. In contrast, the -90 to -46 region by itself had little activity but it played an accessory role by increasing transcriptional activity of the two distal regions.
Although, the CaMV 35S promoter is a strong promoter, driving high levels of RNA production in a wide variety of plants including plants well outside the host range of the virus, it has relatively low activity in the agriculturally significant graminaceous plants such as wheat (Lee et al. (1987) in "Progress in Plant Protoplast Research," Proceedings of the 7th International Protoplast Symposium, Wageningen, The Netherlands, Dec. 6-11, 1987, Puite et al. (eds.); Hauptmann et al. (1987) Plant Cell Rep. 6:265-270). Conversely, the monocot promoter from the Adh1 gene of maize gives very low expression in protoplasts of the dicot, Nicotiana plumbaginifolia (Ellis et al. (1987) EMBO J. 6:11-16). These observations suggest that there may be differences between monocots and dicots with respect to transcription factors and the recognition of promoter sequences.
The level of expression of a transgene can often be increased by the addition of enhancer elements, cis-acting sequences which increase the level of transcription from a promoter (Banerji et al. (1981) Cell 27:299-308). As defined by Khoury and Gruss (1983) Cell 33:313-314, an enhancer is one of a set of eukaryotic promoter elements that appears to increase transcriptional efficiency in a manner relatively independent of position and orientation with respect to the nearby gene. The prototype enhancer is found within the 72 bp repeat of SV40. It is located more than 100 bp upstream from the transcription start site, and has a consensus sequence of GTGGAAA(orTTT)G. As a rule the animal or animal virus enhancers can act over a distance as much as 1 kbp 5', in either orientation, and can act either 5' or 3' to the gene. The sequence motif is generally reiterated several times. Enhancers have been used in animal virus systems to study genes with weak promoters (Lee et al. (1981) Nature 294:228-232; Huang et al. (1981) Cell 27:245-255). There have been sequences from plant genes described which have homology to the animal enhancer consensus core sequence. Odell et al. (1985) Nature 313:810-812 have shown that sequences between -105 and -46 are required for maximal expression of the CaMV 35S promoter. Contained within that region is a sequence partially homologous to the animal enhancer core consensus sequence. It has been shown further by Bouchez et al. (1989) EMBO J. 8:4197-4204 that a 31 bp fragment from -89 to -59 of the 35S promoter contains a binding site for a nuclear protein factor present in maize and tobacco nuclei (Singh al. (1989) Proc. Natl. Acad. Sci. 86:3733-3737) and is essential for maximal activity of the promoter. Similar enhancer sequences have been found in upstream regions of the figwort mosaic virus (FMV), the carnation etched ring virus (CERV), and of seven T-DNA opine synthase genes from Ri and Ti plasmids. Ellis et al. (1987) EMBO J. 6:11-16 have shown that deletion of upstream sequences of the Adh1 promoter (from positions -1094 to -140) gave an Adh1 gene construct having only extremely low expression in transgenic tobacco. However, activity was readily detected when sequences with enhancer-like properties derived from two constitutive genes, octopine synthase (ocs) and the CaMV 35S gene, which are expressed in dicot plants, are placed upstream of the maize Adh1 promoter region. It was shown that the first 247 bp of sequence upstream of the translation initiation codon of the maize Adh1 gene confers anaerobic regulation and accurate transcription initiation to the hybrid gene in transgenic tobacco. It was further shown (Ellis et al. (1987) EMBO J. 6:3203-3208) that a 176 bp DNA sequence derived from the upstream region of the ocs promoter functions as an enhancer in protoplasts of Zea mays, a monocot plant, and Nicotiana plumbaginifolia, a dicot plant. This 176 ocs sequence was reported to function in both orientations, but its enhancing activity was found to be dependent upon its distance from the Adh1 promoter, and also to result from the presence of a 16 bp palindrome having the sequence ACGTAAGCGCTTACGT (SEQ ID NO:6).
In other studies Kay et al. (1987) Science 236:1299-1302 reported a ten-fold higher transcriptional activity in transgenic tobacco plants with a CaMV 35S promoter containing a duplication of 260 bp of CaMV 35S upstream sequences. The duplicated region was reported also to act as a strong enhancer of heterologous promoters, increasing the activity of an adjacent and divergently transcribed transferred DNA gene several hundred fold.
It was also reported by Ow et al. (1987) Proc. Natl Acad. Sci. 84:4870-4874 that multimers of the distal region of the 35S promoter (between positions -148 and -89) were able to activate the 35S promoter core to even greater levels of expression than the native 35S promoter. It was further reported by Fang et al. (1989) supra that monomers and multiples of an upstream 35S promoter fragment (-209 to -46) can act as enhancers to potentiate transcription from a heterologous promoter. In these studies eight copies of the upstream region between positions -209 to -46 of the 35S promoter were cloned at position -50 of the rbcS-3A (small subunit of the ribulose bisphosphate carboxylase) gene; the octamer increased the rbcS-3A transcript to a level even higher than that obtained with the rbcS-3A upstream region (Fang et al. (1989) supra).
Enhancers obtained from sources such as viral or bacterial genomes were shown to function in enhancement of expression in plants of a desired gene. In one such case, the species-specificity of a promoter was modified by the addition of the octopine synthase (OCS) enhancer from Agrobacterium tumefaciens to the maize Adh1 promoter (Ellis et al. (1987) EMBO J. 6:11-16). After addition of the OCS enhancer, the maize Adh1 promoter is able to give strong anaerobically inducible expression in transgenic tobacco plants. In another case, it was reported that when the OCS enhancer is placed directly adjacent to the ARE, the OCS-ARE construct shows maximal expression in maize protoplasts and CAT expression is not further increased by anaerobic stress (Peacock et al. (1987) in Plant Gene Systems and Their Biology, Alan R. Liss, Inc., pp. 263-277). It was also reported (Callis et. al. (1987) Genes and Dev. 1:1183-1200) that the inclusion of the maize Adh1 Intron 1 downstream of the Adh1 promoter in the untranslated leader has been shown to increase expression ten-fold from a chloramphenicol acetyltransferase (CAT) marker gene introduced into maize protoplasts.